The present invention relates to a polypeptide which can prevent avian infectious coryza. More particularly, the present invention relates to a polypeptide from Haemophilus paragallinarum, the causative agent of avian infectious coryza, a gene coding for said polypeptide and an antibody protein which recognizes said polypeptide. The present invention further relates to a process for preparing said polypeptide and the use of said polypeptide for a vaccine, a diagnostic agent and a therapeutic agent.
Avian infectious coryza is one of the most important respiratory diseases in poultry, which is an acute respiratory disease caused by infection with Haemophilus paragallinarum (hereinafter also referred to as xe2x80x9cHPGxe2x80x9d) with cardinal symptoms being a running nose, swelling of the face and epiphora. Avian infectious coryza brings about a great economical damage since it leads to decrease in the breeding rate of poultry, retarding of egg laying, decrease in egg production or failure of egg laying. For prevention of avian infectious coryza, an inactivated vaccine has hitherto been used widely which is obtained by culturing Haemophilus paragalinarum, recovering and inactivating the cells with formalin, thimerosal and the like. However, adverse side effects caused by such an inactivated vaccine has been an issue as it has been reported that local necrotic lesions are formed in the inoculated chicken when the vaccine is administered (M. Matsumoto and R. Yamamoto, Avian Dis., 15: 109-117, 1971), and hence, development of a highly safe vaccine is earnestly desired.
In recent years, laborsaving in breeding and managing poultry is in progress with a scale-up of breeding poultry. As a part of this, laborsaving in vaccination has also been earnestly desired, and as a result, a mixed vaccine has already been developed and widely used in the field so that a frequency of inoculation can be reduced by mixing several kinds of vaccines together.
In order to provide a mixed vaccine showing immunogenicity equivalent to that of each plain vaccine without increase of dosage amount, it is necessary to increase an amount of each antigen contained in a mixed vaccine or to find out and use a more suitable adjuvant. However, in case of gram-negative bacteria such as HPG, a higher amount of antigen is likely to enhance a response to injection such as swelling at the inoculated site. Therefore, in order to reduce such an adverse response, it is preferable to obtain only a protective antigen, i.e. an effective component, from bacterial cells or culture supernatant, or to clone a gene coding for said antigen by the genetic recombination technique, to express said gene in bacteria, yeast, an animal cell, a plant cell, an insect cell and the like, and to purify a product expressed in a large amount, which is then mixed with an appropriate adjuvant together with other vaccines.
Another approach for laborsaving of vaccination is the use of virus or bacteria as a vector. That is, genes coding for protective antigens from one or plural pathogens have been incorporated into an attenuated virus or bacteria to prepare a polyvalent live vaccine. For fowls, poxvirus, Marek""s disease virus and the like have been investigated as a vector. A vaccine comprising a viral vector has been put into practice wherein genes coding for HN and F proteins of Newcastle disease virus are incorporated into fowl pox virus.
It is thus most important to identify a protective antigen of HPG for development of a safe and effective vaccine against avian infectious coryza both as a component vaccine and as a vector vaccine.
Among protective antigens of HPG such as hemagglutinin (HA) and outer-membrane protein, HA is considered a most important antigen since immunization of chicken with HPG increases a hemagglutination-inhibition antibody (hereinafter referred to as xe2x80x9cHI antibodyxe2x80x9d) and higher protective effect is observed for chickens with high level of HI antibody (K. Otsuki and Y. Iritani, Avian Dis., 18: 297-304, 1974 and K. Kume et al., Jpn. J. Vet. Sci., 46: 843-850, 1984).
Serotype of HPG is classified into serotypes A, B and C (Page, Am. J. Vet. Res., 23: 85-95, 1962) or into serotypes 1 and 2 (Sawata et al., Jpn. J. Vet. Sci., 40: 645-652, 1978) based on the agglutination test. It is considered that serotype A by Page corresponds to serotype 1 by Sawata et al. whereas serotype C by Page corresponds to serotype 2 by Sawata et al. (K. Kume, et al., Am. J. Vet. Res., 41: 757-760, 1980 and Sawata et al., Am. J. Vet. Res., 41: 1901-1904, 1980).
Kume et al. reported that HPG serotype A (serotype 1) has at least three kinds of HA, i.e. HA-L (heat-labile, trypsin-sensitive), HA-HL (heat-labile, trypsin-resistant) and HA-HS (heast-stable, trypsin-resistant), and that HA-L alone exhibits not only HA activity to usual fresh chicken erythrcytes but also to glutaraldehyde-fixed chicken erythrocytes and is involved in protection against infection with HPG serotype A (K. Kume, Jpn. J. Vet. Sci., 45: 783-792, 1983 and Sawata et al., Jpn. J. Vet. Sci., 46: 21-29, 1984).
Iritani et al. reported that HPG serotype A has two kinds of HA, i.e. type 1 HA (heat-labile, protease-sensitive) and type 2 HA (heat-labile, protease-resistant), and that type 1 HA, which is heat-labile and protease-sensitive and consisted of a polypeptide having a molecular weight of about 39 kd as a subunit, is involved in protection against infection (T. Yamaguchi and Y Iritani, Jpn. J. Vet. Sci., 42: 709-711, 1980 and Y. Iritani et al., Am. J. Vet. Res., 41: 2114-2118, 1980). It is considered that HA-L and HA-HL by Kume et al. correspond to type 1 HA and type 2 HA by Iritani et al., respectively. As to HPG serotype C (serotype 2), Sawata et al. reported that an antigen was found which is heat-labile and trypsin-sensitive and exhibits the HA activity to glutaraldehyde-fixed chicken erythrocytes and that this antigen is distinct from HA of HPG serotype A in their antigenicity (Sawata et al., Am. J. Vet. Res., 43: 1311-1314, 1982). However, to date, a protective antigen of HPG has not yet seen materially identified except for type 1 HA produced by HPG serotype A as reported by Iritani et al.
As mentioned hereinabove, the conventional inactivated vaccine obtained by inactivating Haemophilus paragallinarum cells with thimerosal, formalin and the like has provoked problems in that the adverse side effects as mentioned above are induced when it is applied to fowls in a large amount since it includes various substances from the cells other than the protective antigen.
The inventor has earnestly studied in order to solve the problems, and as a result, has successfully purified, from a culture supernatant of Haemophilus paragallinarum serotype A, a polypeptide having about 130 kd of molecular weight from Haemophilus paragallinarum serotype A, said polypeptide inducing production of HI antibody and protecting against avian infectious coryza by Haemophilus paragallinarum serotype A.
Furthermore, the present inventor has prepared a genomic DNA library from HPG serotype A, cloned a gene fragment coding for the above 130 Kd polypeptide, expressed said gene fragment in E. coli and has found that the produced polypeptide could prevent avian infectious coryza by Haemophilus paragallinarum serotype A. Said gene fragment coding for the above 130 Kd polypeptide was also used as a probe for cloning a gene fragment hybridizable with said DNA fragment from HPG serotype C to give E. coli which expresses the polypeptide from HPG serotype C.
The present invention provides a safer, effective vaccine against avian infectious coryza, pathogenic bacteria of which is Haemophilus paragallinarum, with less adverse side effects and a process for preparing the same.
That is, an object of the present invention is to provide a novel polypeptide from Haemophilus paragallinarum as well as a peptide which shares at least a portion of the amino acid sequence.
Another object of the present invention is to provide a gene coding for said novel polypeptide from Haemophilus paragallinarum as well as the peptide which shares at least a potion of the amino acid sequence and a recombinant vector for expression of said gene.
Still another object of the present invention is to provide a process for preparing said novel polypeptide from Haemophilus paragallinarum and the polypeptide which shares at least a portion of the amino acid sequence from microorganisms or cells transformed with said recombinant vector.
Still further object of the present invention is to provide a monoclonal or polyclonal antibody which is prepared by using as an immunogen the thus prepared novel peptide from Haemophilus paragallinarum or the polypeptide which shares at least a portion of the amino acid sequence.
Still another object of the present invention is to provide a method for detecting Haemophilus paragallinarum or an antibody thereto by a combination of the above-mentioned peptide, DNA fragment, transformant or antibody.
Still further object of the present invention is to provide a therapeutic agent for avian infectious coryza which comprises as an active ingredient the antibody against the novel polypeptide from Haemophilus paragallinarum.